Tyrosine kinases are a subgroup of the larger class of protein kinases and they function to transfer a phosphate group from ATP to a tyrosine residue in a protein. Phosphorylation of proteins by kinases is an important mechanism in signal transduction for regulating cell metabolism. Dysregulation of tyrosine kinase activity and expression is implicated in several diseases, which makes these enzymes valuable drug targets for the pharmaceutical industry. It is thus desirable to develop assays for the detection of their activity in cells and other biological systems and samples.
While most tyrosine kinases have known specific substrates, it is convenient for in vitro kinase assays to use a general purpose substrate that can be recognized by more than one tyrosine kinase. Poly-(Glu-Ala-Tyr), also known as poly-GAT or poly-EAY, and poly-(Glu, Tyr), also known as poly-GT or poly-EY, are heterogeneous co-polymer substrates that can be phosphorylated by a variety of tyrosine kinases.
Poly-(Glu-Ala-Tyr) is a copolymer made by the random polymerization of glutamic acid (E), alanine (A) and tyrosine (Y) in various molar ratios. Poly-(Glu-Ala-Tyr) is commercially available (e.g. Sigma-Aldrich Co.) as random copolymers 1:1:1 and 6:3:1 ratios. Poly-(Glu, Tyr) is a random co-polymer of glutamic acid and tyrosine, usually in a 4:1 ratio. Both types of polymer are heterogeneous in size, with molecular weights ranging from 20,000 to 50,000 Da.
The heterogeneity of these mixtures of random copolymers, poly-EY and poly-EAY, in many assay formats raises problems of variability in labeling with dyes, fluorophores or tag groups, and/or performance in kinase assays. In addition, these heterogeneous mixtures of random co-polymers have limited stability in solution. Once dissolved in aqueous buffer, both poly-EY and poly-EAY have to be kept, even for short term storage, at −80° C. to preserve their functionality as kinase substrates. Further, these heterogeneous mixtures of random co-polymers have limited stability in particular kinase assays, such as time-resolved fluorescence energy transfer (TR-FRET) kinase assays.
There is a continuing need for stable, homogeneous, generic peptide substrates phosphorylated by multiple tyrosine kinases.